Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Abstract

28Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis 29 occurs, and humans where parasites proliferate asexually. We describe a powerful approach to 30 identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-31 cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis 32 (BSA). We combined experimental genetic crosses using humanized mice, with selective whole 33 genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate 34 host to examine parasite competition and identify genomic regions under selection. We generated 35 crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, 36 kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified 37 genome-wide changes in allele frequency in the parasite progeny population from infected midgut 38 and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and 39 aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results 40 emerge: we observed (i) a strong skew (>80%) towards alleles from the ART-R parent in the 41 mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected 42 against; (ii) highly repeatable skews in allele frequencies across the genome in blood stage 43 parasites; (iii) particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against 44 alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 45 containing ARPS10. This approach robustly identifies selected loci and has strong potential for 46 identifying parasite genes that interact with the mosquito vector or compensatory loci involved in 47 drug resistance. 48 49 3 50 Parasitic organisms frequently use multiple hosts and have several morphologically and 51 transcriptionally distinctive life cycle stages. Within each host, parasites must circumvent immune 52 defenses and navigate to new tissues. There are frequently extreme bottlenecks in parasite numbers 53 during transmission (Hopp et al. 2015), with rapid proliferative growth within hosts, and intense 54 competition between co-infecting parasite genotypes. For example, the life cycle of malaria 55 parasites involves successive infection of two hosts: female Anopheles mosquitoes, where gamete 56 fusion, meiosis and recombination occurs, and humans in which parasites travel from the skin, 57 develop in the liver and then proliferate asexually in the blood stream. Ideally, we would like to 58 understand how natural selection operates across the complete life cycle and document the genes 59 subject to selection pressures at each life cycle stage: during erythrocytic growth, gametocyte 60 production, oocyst development in the mosquito midgut, migration of sporozoites to the salivary 61 glands, transmission from the salivary glands, sporozoite survival in the skin, and est...

Bulk segregant analysis reveals environment × genotype interactions determining malaria parasite growth

“…We observed three genome regions that showed strong distortions in allele frequency in each independent replicate cross in both human serum and AlbuMAX cultures ( Fig. 2A), consistent with our earlier report (14,15). We used G' values to measure the significance of these allelic skews ( Fig.…”

“…14. ARPS10 alleles with these two mutations (NHP4026 in this study and NHP1337 in previous study by Li et al (15)) carry a high fitness cost during in vitro culture with serum. The Val127Met mutation is one of the genetic background SNPs for kelch13 alleles on which artemisinin resistance emerged in Cambodia (22).…”

“…We generated the crosses using Anopheles stephensi mosquitoes and FRG huHep mice as described in Vaughan et al (13) and Li et al (15). P. falciparum parasites NF54 and NHP4026 were cloned by limiting dilution and used as parents for this cross.…”

“…14 (numbered 14.2 in Table S5) spanned from 2,356 kb to 2,485 kb (129 kb, 23 genes, 10 high priority candidates). These same regions carried high fitness costs in a BSA analysis from an independent genetic cross between two different parental parasites -ART-S MKK2835 and ART-R NHP1337 (15). The multidrug resistance-associated protein 2 (MRP2, PF3D7_1229100) and apicoplast ribosomal protein S10 (ARPS10, PF3D7_1460900), are both located inside of the chr.…”

“…The FRG huHep mouse supports liver stage development of P. falciparum and the transition to asexual blood stage. We have previously generated four independent P. falciparum genetic crosses with large numbers of unique recombinant progeny using this approach (13)(14)(15).…”